Human cytomegalovirus (HCMV) infects over 60% of the adult population. It is a major cause of birth defects, and a life-threatening opportunistic agent in immunosuppressed people. Congenital infection has a significant prevalence and can cause permanent disabilities. Proteomic analysis in our lab has recently revealed that numerous cell-surface proteins are dynamically modulated following HCMV infection of fibroblasts, including 27 cell-surface adhesion proteins. Two upregulated adhesion proteins, L1CAM and GPR56, have been tested further and shown to significantly impact the yield of HCMV progeny. These observations form the foundation for the central hypothesis underlying this application: multiple cellular adhesion proteins cooperate to modulate the outcome of HCMV infection. To address this hypothesis, we will first study the IgCAM family member, L1CAM, which is elevated in a wide range of human tumors, inducing radioresistance and often enhancing tumor progression or metastasis. Our preliminary studies show that cell-surface L1CAM is elevated by infection and supports the production of HCMV progeny in fibroblasts. Secreted L1CAM extracellular domain (L1CAM-ECD, sponsors autocrine and paracrine signal transduction) and nuclear L1CAM intracellular domain (L1CAM-ICD modulates cellular gene expression) also are dramatically increased following infection. Further, knockdown of L1CAM sensitized infected cells to radiomimetic drugs. The remarkable range of activities documented for L1CAM provide a compelling rationale for detailed study of its role in HCMV replication and spread, i.e., L1CAM is likely to impact HCMV replication and spread at multiple levels: a direct effect on the production of HCMV progeny; and possible effects on viral spread via infected-cell migration, the infected-cell microenvironment via L1CAM-ECD activity, and the radiosensitivity/DNA-damage response of infected cells. We also will investigate GPR56, a member of the adhesion family of G protein-coupled receptors. Our initial studies of the HCMV-infected cell proteome used fibroblasts, which are infected at numerous sites within the body and serve as a reference, since they are the common laboratory host cell for HCMV studies. We will next characterize epithelial cells to expand our understanding of the infected cell-surface proteome and identify further candidates for detailed studies such as those proposed above for L1CAM. Epithelial cells are central to HCMV pathogenesis, because they are infected at the site of entry, they host long-term low-level replication in the salivary gland, breast and kidney to mediate HCMV spread to new hosts, and they are the target cell in CMV retinitis. Collectively, this study will provide he first broad-based, mechanistic characterization of adhesion proteins during HCMV infection. Successful completion of these studies should contribute importantly to the understanding of a relatively understudied area of HCMV biology, the role of cellular adhesion proteins in viral replication and dissemination.